MULTITARGET DRUGS: VERIFICATION-OF-PRINCIPLE STUDIES

药物选择性不足是当今治疗方法的一大弊病。这个问题对抗菌药物来说意义重大，对抗病毒药物来说困难，而对抗癌药物来说则完全没有解决。抗癌药物对主要癌症仍然无效，而且还会产生严重的副作用。如果治疗剂能够获得多靶点、组合选择性，杀死（或以其他方式修饰）一个细胞，当且仅当它包含一组预定的分子靶标而缺乏另一组预定的靶标时，这个问题可能得到解决。最近，提出了一种适用于小（小于或等于1kd）化合物的药物选择性问题的解决方案（Varshavsky, a . PNAS 95, 2094-2099(1998)）。两个概念，干扰/共优势（IC）和抗效应物，应该允许治疗方案具有组合选择性，其中阳性和阴性感知的大分子靶标的数量可以是两个，三个或更多。这些药物的体内作用类似于布尔运算“和”、“或”非”及其组合的结果。拟议的研究将得到当前R21申请的支持，将验证以酵母酿酒酵母作为模式生物的多靶点设计原则。1)泛素融合技术将用于在酵母酵母中表达和分析一组多片段线性肽，这些肽在体内环境中作为IC型化合物。2)将合成由无水四环素（at）片段和甲氨蝶呤（mtx）或三甲蝶呤（tm）片段连接at组成的双组分ic型化合物，然后在体外（与纯化的TetR或DHFR结合的互他性）和体内（对缺乏或表达at结合的大肠杆菌TetR抑制因子的葡萄球菌的差异毒性）进行表征。3)合成含有新生物素（nov）片段和甲氨蝶呤（mtx）或三甲氨蝶呤（tm）片段的双组分ic型化合物，并对缺乏或表达大肠杆菌DNA旋转体（GyrB）的非结合b亚基的cerevisiae进行差异细胞毒性测试。4)合成含有at片段、β -脱皮素（ec）片段和甲氨蝶呤（mtx）或三甲蝶呤（tm）片段的ic型三元化合物，并对缺乏或表达相应大分子靶标及其成对组合的酵母进行差异毒性测试。

The insufficient selectivity of drugs is a bane of present-day therapies. This problem is significant for antibacterial drugs, difficult for antivirals, and utterly unsolved for anti-cancer drugs, which remain ineffective against major cancers, and in addition cause severe side effects. T he problem may be solved if therapeutic agents could acquire a multi-target, combinatorial selectivity, killing (or otherwise modifying) a cell if, and only if, it contains a predetermined set of molecular targets and lacks another predetermined set of targets. Recently, a solution to the problem of drug selectivity was proposed that is applicable to small (less than or equal to 1 kD) compounds (Varshavsky, A. PNAS 95, 2094-2099 (1998)). Two ideas, interference/co-dominance (IC) and anti-effectors, should allow a therapeutic regimen to possess combinatorial selectivity, in which the number of positively and negatively sensed macromolecular targets can be two, three, or more. The in vivo effects of such drugs would be analogous to the outcomes of the Boolean operations "and", "or" "not", and combinations thereof. The proposed studies, to be supported by the present R21 application, will verify the principles of multi-target designs using the yeast Saccharomyces cerevisiae as a model organism. Specific Aims 1) The ubiquitin fusion technique will be used to express and analyze, in the yeast S. cerevisiae, a set of multi-moiety linear peptides that act as IC- type compounds in this in vivo setting. 2) Bipartite, IC-type compounds that comprise the anhydrotetracycline (at) moiety and either the methotrexate (mtx) or the trimetrexate (tm) moiety linked to at will be synthesized, and thereafter characterized in vitro (mutual exclusivity of binding to the purified TetR or DHFR) and in vivo (differential toxicity to S. cerevisiae that either lack or express the at- binding E. coli TetR repressor). 3) Bipartite, IC-type compounds containing the novobiocin (nov) moiety and either the methotrexate (mtx) or the trimetrexate (tm) moiety will be synthesized and tested for differential cytotoxicity with S. cerevisiae that either lack or express the nov-binding B-subunit of E. coli DNA gyrates (GyrB). 4) Tripartite, IC-type compounds containing the at moiety, the beta- ecdysone (ec) moiety, and either the methotrexate (mtx) or the trimetrexate (tm) moiety will be synthesized and tested for differential toxicity with S. cerevisiae that lack or express the corresponding macromolecular targets and their pairwise combinations.